Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

Effects of SPI1-mediated transcriptome remodeling on Alzheimer's disease-related phenotypes in mouse models of Aß amyloidosis.

SPI1 was recently reported as a genetic risk factor for Alzheimer's disease (AD) in large-scale genome-wide association studies. However, it is unknown whether SPI1 should be downregulated or increased to have therapeutic benefits. To investigate the effect of modulating SPI1 levels on AD pathogenesis, we performed extensive biochemical, histological, and transcriptomic analyses using both Spi1-knockdown and Spi1-overexpression mouse models. Here, we show that the knockdown of Spi1 expression significantly exacerbates insoluble amyloid-ß (Aß) levels, amyloid plaque deposition, and gliosis. Conversely, overexpression of Spi1 significantly ameliorates these phenotypes and dystrophic neurites. Further mechanistic studies using targeted and single-cell transcriptomics approaches demonstrate that altered Spi1 expression modulates several pathways, such as immune response pathways and complement system. Our data suggest that transcriptional reprogramming by targeting transcription factors, like Spi1, might hold promise as a therapeutic strategy. This approach could potentially expand the current landscape of druggable targets for AD.

Preclinical evaluation of Tc-99m p5+14 peptide for SPECT detection of cardiac amyloidosis.

INTRODUCTION: Amyloid deposition is a cause of restrictive cardiomyopathy. Patients who present with cardiac disease can be evaluated for transthyretin (TTR)-associated cardiac amyloidosis using nuclear imaging with 99mTc-labeled pyrophosphate (PYP); however, light chain-associated (AL) cardiac amyloid is generally not detected using this tracer. As an alternative, the amyloid-binding peptide p5+14 radiolabeled with iodine-124 has been shown to be an effective pan-amyloid radiotracer for PET/CT imaging. Here, a 99mTc-labeled form of p5+14 peptide has been prepared to facilitate SPECT/CT imaging of cardiac amyloidosis. METHOD: A synthesis method suitable for clinical applications has been used to prepare 99mTc-labeled p5+14 and tested for peptide purity, product bioactivity, radiochemical purity and stability. The product was compared with99mTc-PYP for cardiac SPECT/CT imaging in a mouse model of AA amyloidosis and for reactivity with human tissue sections from AL and TTR patients. RESULTS: The 99mTc p5+14 tracer was produced with >95% yields in radiopurity and bioactivity with no purification steps required and retained over 95% peptide purity and >90% bioactivity for >3 h. In mice, the tracer detected hepatosplenic AA amyloid as well as heart deposits with uptake ~5 fold higher than 99mTc-PYP. 99mTc p5+14 effectively bound human amyloid deposits in the liver, kidney and both AL- and ATTR cardiac amyloid in tissue sections in which 99mTc-PYP binding was not detectable. CONCLUSION: 99mTc-p5+14 was prepared in minutes in >20 mCi doses with good performance in preclinical studies making it suitable for clinical SPECT/CT imaging of cardiac amyloidosis.

[New Development in Amyloidosis Research Based on Supersaturation: Biological Factors to Induce/Inhibit the Amyloid Fibril Formation].

Amyloid fibril formation is a general property of proteins and peptides. It is a physicochemical phenomenon similar to crystallization, in which amyloid precursor proteins exceeding solubility precipitate through the breakdown of supersaturation. Using the ultrasonication-forced amyloid fibril inducer HANABI, we have discovered that serum albumin acts as an inhibitor in dialysis-related amyloidosis. Exploring the factors that induce or inhibit amyloid fibril formation using HANABI can lead to the development of early diagnosis and prevention methods for amyloidosis.

Cardiac amyloid deposition and the forensic autopsy - A review and analysis.

Although amyloid material in the heart is not infrequently encountered at autopsy it may on occasion be difficult to determine the significance in terms of possible contributions to the terminal mechanisms of death. A review was undertaken of the literature and of autopsy cases at Forensic Science SA over a 20-year-period (2003-2022) for all cases where significant amyloid material had been encountered on microscopy of the heart. Sixteen cases were found consisting of 11 cases where cardiac amyloid was involved in the lethal episode, and five where it was considered an incidental feature. Of the 11 lethal cases, there were three where cardiac amyloidosis was the cause of death, and eight where it was a contributing factor, along with ischaemic heart disease (N = 7) and bronchopneumonia (N = 1). The age range was 47-92 years, average 78.6 years, with a male to female ratio of 10:1. The weights of the hearts ranged from 496 to 1059 g - average 648 g. Of the five cases where it was considered an incidental finding, the causes of death were blunt head trauma (N = 2), small intestinal ischaemia (N = 2) and small intestinal obstruction (N = 1). The weights of the hearts ranged from 299 to 487 g, average 369 g. The most relevant types of amyloidosis in forensic cases tend to be light chain amyloidosis, senile cardiac amyloidosis and familial amyloid cardiomyopathy. Other forms of amyloidosis that affect the heart, which include reactive amyloidosis, haemodialysis-related amyloidosis and isolated atrial amyloidosis, either have minimal or no clinical significance, or are of uncertain significance. While it may be difficult to determine the prognostic significance of amyloid material at autopsy clinicopathological correlation may provide useful supportive information.

Biophysical insight into anti-amyloidogenic nature of novel ionic Co(II)(phen)(H2O)4]+[glycinate]- chemotherapeutic drug candidate against human lysozyme aggregation.

In the recent past, there has been an ever-increasing interest in the search for metal-based therapeutic drug candidates for protein misfolding disorders (PMDs) particularly neurodegenerative disorders such as Alzheimer's, Parkinson's, Prion's diseases, and amyotrophic lateral sclerosis. Also, different amyloidogenic variants of human lysozyme (HL) are involved in hereditary systemic amyloidosis. Metallo-therapeutic agents are extensively studied as antitumor agents, however, they are relatively unexplored for the treatment of non-neuropathic amyloidoses. In this work, inhibition potential of a novel ionic cobalt(II) therapeutic agent (CoTA) of the formulation [Co(phen)(H2O)4]+[glycinate]- is evaluated against HL fibrillation. Various biophysical techniques viz., dye-binding assays, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electron microscopy, and molecular docking experiments validate the proposed mechanism of inhibition of HL fibrillation by CoTA. The experimental corroborative results of these studies reveal that CoTA can suppress and slow down HL fibrillation at physiological temperature and pH. DLS and 1-anilino-8-naphthalenesulfonate (ANS) assay show that reduced fibrillation in the presence of CoTA is marked by a significant decrease in the size and hydrophobicity of the aggregates. Fluorescence quenching and molecular docking results demonstrate that CoTA binds moderately to the aggregation-prone region of HL (Kb = 6.6 × 104 M-1), thereby, inhibiting HL fibrillation. In addition, far-UV CD and DSC show that binding of CoTA to HL does not cause any change in the stability of HL. More importantly, CoTA attenuates membrane damaging effects of HL aggregates against RBCs. This study identifies inorganic metal complexes as a therapeutic intervention for systemic amyloidosis.

Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer's disease mice.

BACKGROUND: Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer's disease (AD) if these macromolecules can cross the blood-brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD). METHODS: Eight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics. RESULTS: We observed regional differences in proteins relevant to Aß, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice. From 64 target proteins studied using spatial proteomics, a comparison of the Aß-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aß and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (⁓ 33%). TfRMAb-TNFR significantly reduced mature Aß plaques and increased Aß-associated microglia around larger Aß deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aß plaque-associated microglial TREM2 in 3xTg-AD mice. CONCLUSION: Overall, despite the low visual Aß load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.

Cardiac Amyloidosis Due to Transthyretin Protein: A Review.

Importance: Systemic amyloidosis from transthyretin (ATTR) protein is the most common type of amyloidosis that causes cardiomyopathy. Observations: Transthyretin (TTR) protein transports thyroxine (thyroid hormone) and retinol (vitamin A) and is synthesized predominantly by the liver. When the TTR protein misfolds, it can form amyloid fibrils that deposit in the heart causing heart failure, heart conduction block, or arrhythmia such as atrial fibrillation. The biological processes by which amyloid fibrils form are incompletely understood but are associated with aging and, in some patients, affected by inherited variants in the TTR genetic sequence. ATTR amyloidosis results from misfolded TTR protein deposition. ATTR can occur in association with normal TTR genetic sequence (wild-type ATTR) or with abnormal TTR genetic sequence (variant ATTR). Wild-type ATTR primarily manifests as cardiomyopathy while ATTR due to a genetic variant manifests as cardiomyopathy and/or polyneuropathy. Approximately 50 000 to 150 000 people in the US have heart failure due to ATTR amyloidosis. Without treatment, heart failure due to ATTR amyloidosis is associated with a median survival of approximately 5 years. More than 130 different inherited genetic variants in TTR exist. The most common genetic variant is Val122Ile (pV142I), an allele with an origin in West African countries, that is present in 3.4% of African American individuals in the US or approximately 1.5 million persons. The diagnosis can be made using serum free light chain assay and immunofixation electrophoresis to exclude light chain amyloidosis combined with cardiac nuclear scintigraphy to detect radiotracer uptake in a pattern consistent with amyloidosis. Loop diuretics, such as furosemide, torsemide, and bumetanide, are the primary treatment for fluid overload and symptomatic relief of patients with ATTR heart failure. An ATTR-directed therapy that inhibited misfolding of the TTR protein (tafamidis, a protein stabilizer), compared with placebo, reduced mortality from 42.9% to 29.5%, reduced hospitalizations from 0.7/year to 0.48/year, and was most effective when administered early in disease course. Conclusions and Relevance: ATTR amyloidosis causes cardiomyopathy in up to approximately 150 000 people in the US and tafamidis is the only currently approved therapy. Tafamidis slowed progression of ATTR amyloidosis and improved survival and prevented hospitalization, compared with placebo, in people with ATTR-associated cardiomyopathy.

Amyloid-induced neurodegeneration: A comprehensive review through aggregomics perception of proteins in health and pathology.

Amyloidosis of protein caused by fibrillation and aggregation are some of the most exciting new edges not only in protein sciences but also in molecular medicines. The present review discusses recent advancements in the field of neurodegenerative diseases and therapeutic applications with ongoing clinical trials, featuring new areas of protein misfolding resulting in aggregation. The endogenous accretion of protein fibrils having fibrillar morphology symbolizes the beginning of neuro-disorders. Prognostic amyloidosis is prominent in numerous degenerative infections such as Alzheimer's and Parkinson's disease, Amyotrophic lateral sclerosis (ALS), etc. However, the molecular basis determining the intracellular or extracellular evidence of aggregates, playing a significant role as a causative factor in neurodegeneration is still unclear. Structural conversions and protein self-assembly resulting in the formation of amyloid oligomers and fibrils are important events in the pathophysiology of the disease. This comprehensive review sheds light on the evolving landscape of potential treatment modalities, highlighting the ongoing clinical trials and the potential socio-economic impact of novel therapeutic interventions in the realm of neurodegenerative diseases. Furthermore, many drugs are undergoing different levels of clinical trials that would certainly help in treating these disorders and will surely improve the socio-impact of human life.

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Diagnosis and prognosis of abnormal cardiac scintigraphy uptake suggestive of cardiac amyloidosis using artificial intelligence: a retrospective, international, multicentre, cross-tracer development and validation study.

BACKGROUND: The diagnosis of cardiac amyloidosis can be established non-invasively by scintigraphy using bone-avid tracers, but visual assessment is subjective and can lead to misdiagnosis. We aimed to develop and validate an artificial intelligence (AI) system for standardised and reliable screening of cardiac amyloidosis-suggestive uptake and assess its prognostic value, using a multinational database of 99mTc-scintigraphy data across multiple tracers and scanners. METHODS: In this retrospective, international, multicentre, cross-tracer development and validation study, 16 241 patients with 19 401 scans were included from nine centres: one hospital in Austria (consecutive recruitment Jan 4, 2010, to Aug 19, 2020), five hospital sites in London, UK (consecutive recruitment Oct 1, 2014, to Sept 29, 2022), two centres in China (selected scans from Jan 1, 2021, to Oct 31, 2022), and one centre in Italy (selected scans from Jan 1, 2011, to May 23, 2023). The dataset included all patients referred to whole-body 99mTc-scintigraphy with an anterior view and all 99mTc-labelled tracers currently used to identify cardiac amyloidosis-suggestive uptake. Exclusion criteria were image acquisition at less than 2 h (99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid, 99mTc-hydroxymethylene diphosphonate, and 99mTc-methylene diphosphonate) or less than 1 h (99mTc-pyrophosphate) after tracer injection and if patients' imaging and clinical data could not be linked. Ground truth annotation was derived from centralised core-lab consensus reading of at least three independent experts (CN, TT-W, and JN). An AI system for detection of cardiac amyloidosis-associated high-grade cardiac tracer uptake was developed using data from one centre (Austria) and independently validated in the remaining centres. A multicase, multireader study and a medical algorithmic audit were conducted to assess clinician performance compared with AI and to evaluate and correct failure modes. The system's prognostic value in predicting mortality was tested in the consecutively recruited cohorts using cox proportional hazards models for each cohort individually and for the combined cohorts. FINDINGS: The prevalence of cases positive for cardiac amyloidosis-suggestive uptake was 142 (2%) of 9176 patients in the Austrian, 125 (2%) of 6763 patients in the UK, 63 (62%) of 102 patients in the Chinese, and 103 (52%) of 200 patients in the Italian cohorts. In the Austrian cohort, cross-validation performance showed an area under the curve (AUC) of 1·000 (95% CI 1·000-1·000). Independent validation yielded AUCs of 0·997 (0·993-0·999) for the UK, 0·925 (0·871-0·971) for the Chinese, and 1·000 (0·999-1·000) for the Italian cohorts. In the multicase multireader study, five physicians disagreed in 22 (11%) of 200 cases (Fleiss' kappa 0·89), with a mean AUC of 0·946 (95% CI 0·924-0·967), which was inferior to AI (AUC 0·997 [0·991-1·000], p=0·0040). The medical algorithmic audit demonstrated the system's robustness across demographic factors, tracers, scanners, and centres. The AI's predictions were independently prognostic for overall mortality (adjusted hazard ratio 1·44 [95% CI 1·19-1·74], p<0·0001). INTERPRETATION: AI-based screening of cardiac amyloidosis-suggestive uptake in patients undergoing scintigraphy was reliable, eliminated inter-rater variability, and portended prognostic value, with potential implications for identification, referral, and management pathways. FUNDING: Pfizer.

Amyloid ß-Peptide Segment Conjugated Side-Chain Proline-Based Polymers as Potent Inhibitors in Lysozyme Amyloidosis.

Developing effective amyloidosis inhibitors poses a significant challenge due to the dynamic nature of the protein structures, the complex interplay of interfaces in protein-protein interactions, and the irreversible nature of amyloid assembly. The interactions of amyloidogenic polypeptides with other peptides play a pivotal role in modulating amyloidosis and fibril formation. This study presents a novel approach for designing and synthesizing amyloid interaction surfaces using segments derived from the amyloid-promoting sequence of amyloid ß-peptide [VF(Aß(18-19)/FF(Aß(19-20)/LVF(Aß(17-19)/LVFF(Aß(17-20)], where VF, FF, LVF and LVFF stands for valine phenylalanine dipeptide, phenylalanine phenylalanine dipeptide, leucine valine phenylalanine tripeptide and leucine valine phenylalanine phenylalanine tetrapeptide, respectively. These segments are conjugated with side-chain proline-based methacrylate polymers serving as potent lysozyme amyloidosis inhibitors and demonstrating reduced cytotoxicity of amyloid aggregations. Di-, tri-, and tetra-peptide conjugated chain transfer agents (CTAs) were synthesized and used for the reversible addition-fragmentation chain transfer polymerization of tert-butoxycarbonyl (Boc)-proline methacryloyloxyethyl ester (Boc-Pro-HEMA). Deprotection of Boc-groups from the side-chain proline pendants resulted in water-soluble polymers with defined peptide chain ends as peptide-polymer bioconjugates. Among them, the LVFF-conjugated polymer acted as a potent inhibitor with significantly suppressed lysozyme amyloidosis, a finding supported by comprehensive spectroscopic, microscopic, and computational analyses. These results unveil the synergistic effect between the segment-derived amyloid ß-peptide and side-chain proline-based polymers, offering new prospects for targeting lysozyme amyloidosis.

Degradation versus fibrillogenesis, two alternative pathways modulated by seeds and glycosaminoglycans.

The mechanism that converts native human transthyretin into amyloid fibrils in vivo is still a debated and controversial issue. Commonly, non-physiological conditions of pH, temperature, or organic solvents are used in in vitro models of fibrillogenesis of globular proteins. Transthyretin amyloid formation can be achieved under physiological conditions through a mechano-enzymatic mechanism involving specific serine proteases such as trypsin or plasmin. Here, we investigate S52P and L111M transthyretin variants, both causing a severe form of systemic amyloidosis mostly targeting the heart at a relatively young age with heterogeneous phenotype among patients. Our studies on thermodynamics show that both proteins are significantly less stable than other amyloidogenic variants. However, despite a similar thermodynamic stability, L111M variant seems to have enhanced susceptibility to cleavage and a lower tendency to form fibrils than S52P in the presence of specific proteases and biomechanical forces. Heparin strongly enhances the fibrillogenic capacity of L111M transthyretin, but has no effect on the S52P variant. Fibrillar seeds similarly affect the fibrillogenesis of both proteins, with a stronger effect on the L111M variant. According to our model of mechano-enzymatic fibrillogenesis, both full-length and truncated monomers, released after the first cleavage, can enter into fibrillogenesis or degradation pathways. Our findings show that the kinetics of the two processes can be affected by several factors, such as intrinsic amyloidogenicity due to the specific mutations, environmental factors including heparin and fibrillar seeds that significantly accelerate the fibrillogenic pathway.

Spectral Fluorescence Pathology of Protein Misfolding Disorders.

Protein misfolding has been extensively studied in the context of neurodegenerative disorders and systemic amyloidoses. Due to misfolding and aggregation of proteins being highly heterogeneous and generating a variety of structures, a growing body of evidence illustrates numerous ways how the aggregates contribute to progression of diseases such as Alzheimer's disease, Parkinson's disease, and prion disorders. Different misfolded species of the same protein, commonly referred to as strains, appear to play a significant role in shaping the disease clinical phenotype and clinical progression. The distinct toxicity profiles of various misfolded proteins underscore their importance. Current diagnostics struggle to differentiate among these strains early in the disease course. This review explores the potential of spectral fluorescence approaches to illuminate the complexities of protein misfolding pathology and discusses the applications of advanced spectral methods in the detection and characterization of protein misfolding disorders. By examining spectrally variable probes, current data analysis approaches, and important considerations for the use of these techniques, this review aims to provide an overview of the progress made in this field and highlights directions for future research.

Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

Insights into the Structural Basis of Amyloid Resistance Provided by Cryo-EM Structures of AApoAII Amyloid Fibrils.

Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.

The influence of zwitterionic and anionic phospholipids on protein aggregation.

The progressive aggregation of misfolded proteins is the underlying molecular cause of numerous pathologies including Parkinson's disease and injection and transthyretin amyloidosis. A growing body of evidence indicates that protein deposits detected in organs and tissues of patients diagnosed with such pathologies contain fragments of lipid membranes. In vitro experiments also showed that lipid membranes could strongly change the aggregation rate of amyloidogenic proteins, as well as alter the secondary structure and toxicity of oligomers and fibrils formed in their presence. In this review, the effect of large unilamellar vesicles (LUVs) composed of zwitterionic and anionic phospholipids on the aggregation rate of insulin, lysozyme, transthyretin (TTR) and α- synuclein (α-syn) will be discussed. The manuscript will also critically review the most recent findings on the lipid-induced changes in the secondary structure of protein oligomers and fibrils, as well as reveal the extent to which lipids could alter the toxicity of protein aggregates formed in their presence.

Transgenic amyloid precursor protein mouse models of amyloidosis. Incomplete models for Alzheimer's disease but effective predictors of anti-amyloid therapies.

INTRODUCTION: Amyloid precursor protein (APP) transgenic mice are models of Alzheimer's disease (AD) amyloidosis, not all of AD. Diffuse, compacted, and vascular deposits in APP mice mimic those found in AD cases. METHODS: Most interventional studies in APP mice start treatment early in the process of amyloid deposition, consistent with a prevention treatment regimen. Most clinical trials treat patients with established amyloid deposits in a therapeutic treatment regimen. RESULTS: The first treatment to reduce amyloid and cognitive impairment in mice was immunotherapy. The APP mouse models not only predicted efficacy, but presaged the vascular leakage called ARIA. The recent immunotherapy clinical trials that removed amyloid and slowed cognitive decline confirms the utility of these early APP models when used in therapeutic designs. DISCUSSION: New mouse models of AD pathologies will add to the research armamentarium, but the early models have accurately predicted responses to amyloid therapies in humans.

Anakinra-Associated Systemic Amyloidosis.

OBJECTIVE: To describe a 41-year-old woman with a history of neonatal onset multisystem inflammatory disease, on treatment with daily subcutaneous injections of 600 mg of recombinant interleukin-1 receptor antagonist (IL-1Ra) protein, anakinra, since the age of 28, who presented with golf-ball size nodules at the anakinra injection sites, early satiety, new onset nephrotic syndrome in the context of normal markers of systemic inflammation. METHODS: Clinical history and histologic evaluation of biopsies of skin, gastric mucosa, and kidney with Congo-red staining and proteomic evaluation of microdissected Congo red-positive amyloid deposits by liquid chromatography-tandem mass spectrometry. RESULTS: The skin, stomach, and kidney biopsies all showed the presence of Congo red-positive amyloid deposits. Mass spectrometry-based proteomics demonstrated that the amyloid deposits in all sites were of AIL1RAP (IL-1Ra protein)-type. These were characterized by high spectral counts of the amyloid signature proteins (apolipoprotein AIV, apolipoprotein E, and serum amyloid P-component) and the amyloidogenic IL-1Ra protein, which were present in Congo red-positive areas and absent in Congo red-negative areas. The amino acid sequence identified by mass spectrometry confirmed that the amyloid precursor protein was recombinant IL-1Ra (anakinra) and not endogenous wild-type IL-1Ra. CONCLUSION: This is the first report of iatrogenic systemic amyloidosis due to an injectable protein drug, which was caused by recombinant IL1Ra (anakinra).

Early-life stress and amyloidosis in mice share pathogenic pathways involving synaptic mitochondria and lipid metabolism.

INTRODUCTION: Early-life stress (ES) increases the risk for Alzheimer's disease (AD). We and others have shown that ES aggravates amyloid-beta (Aß) pathology and promotes cognitive dysfunction in APP/PS1 mice, but underlying mechanisms remain unclear. METHODS: We studied how ES affects the hippocampal synaptic proteome in wild-type (WT) and APP/PS1 mice at early and late pathological stages, and validated hits using electron microscopy and immunofluorescence. RESULTS: The hippocampal synaptosomes of both ES-exposed WT and early-stage APP/PS1 mice showed a relative decrease in actin dynamics-related proteins and a relative increase in mitochondrial proteins. ES had minimal effects on older WT mice, while strongly affecting the synaptic proteome of advanced stage APP/PS1 mice, particularly the expression of astrocytic and mitochondrial proteins. DISCUSSION: Our data show that ES and amyloidosis share pathogenic pathways involving synaptic mitochondrial dysfunction and lipid metabolism, which may underlie the observed impact of ES on the trajectory of AD.